The present invention relates generally to cross-linked collagen matrices and preparations and more particularly to a novel method for cross linking collagen using reducing sugars and to cross linked collagen matrices and preparations formed by using this method.
Collagens are key molecules of the animal kingdom accounting for approximately 25-30% of all proteins of mammalian organisms. Collagens are natural biopolymers that are organized as fibrillar networks and other forms of superstructures. The fibrillar collagens and particularly type I collagen have the highest incidence accounting for 80% of connective tissues proteins. The high incidence of the fibrillar collagens, their availability from animal sources and the ability to extract and prepare monomeric solutions of purified collagen which can be polymerized into three-dimensional fibrillar matrices make these collagens ideal candidates for natural biomaterials. In addition, the fibrillar collagens exhibit a high degree of conservation and are therefore weak antigens. The main antigenic sites of the fibrillar collagen molecules reside within the non-helical telopeptides which flank the helical portion of the molecule.
In vivo, the polymeric structure of the fibrillar collagens is stabilized by intermolecular cross-links, which are formed by an enzymatic process. Because of the staggered assembly of the collagen molecules, most of these cross-links bridge between the telopeptide domain of one molecule and the helical domain of an adjacent molecule. Additional cross-link formation by the process of glycation takes place as part of the collagen and connective tissues aging.
Glycation of proteins, including collagen, takes place as a physiological process of aging over the life course consequent to the exposure of proteins to glucose. It was found that glycated fibrillar collagens exhibit an increased level of cross-linking and therefore they are more resistant to degradation by collagenases, the specific enzymes which degrade collagen.
The process of glycation by glucose is slow because its physiological concentration in serum is relatively low and only a small proportion of it is found in the acyclic aldehyde form which is the reactive one. It was found that D(xe2x88x92)Ribose is 1000 folds more reactive than glucose in inducing glycation and cross-linking of collagen molecules in fibrillar collagens. For example, incubation of native fibrillar collagen in 0.2 M D(xe2x88x92)ribose for 5 days is equivalent to exposure to physiological concentration of glucose for 20 years. The cross-links produced by glycation bridge form mainly between the triple-helical domains of adjacent molecules.
The performance of collagen-based bioproducts depends on the one hand on controlling their functional longevity within the host and on the other hand on the preservation of the biological properties of the native collagen component. The functional longevity of the collagen component depends on its capacity to resist specific enzymatic degradation by collagenases (metaloproteinases). This capacity is directly related to the number of intramolecular and intermolecular cross-links within the collagen polymer. The higher the number of cross-links the higher the resistance to collagenase degradation.
Exemplary cross-linking agents of choice known in the art have been glutaraldehyde and other related non-physiological agents. These cross-linking agents react with amino acid residues of the collagen molecule to form intermolecular cross-links. However, these harsh agents may have negative effects on the biocompatibility and biological activity of cross-linked collagen-based bioproducts that are caused by alterations in the conformation of the collagen molecule and leaching out of the cross-linking agents. Thus, collagen products cross-linked by non-physiological agents are poorly accepted by and integrated within the host tissues. Furthermore, localized inflammation and more complex systemic reactions are disadvantageous side effects of glutaraldehyde cross-linked collagen products.
U.S. Pat. No. 4,971,954 to Brodsky et al. discloses the use of D(xe2x88x92)Ribose or other reducing physiological sugars as physiological agents for cross-linking collagen matrices by the process of glycation. However, the method disclosed by Brodsky et al. is efficient when the collagenous substrate consists of native collagen fibers, but is only partially effective for collagen matrices produced from reconstituted fibrillar collagen, particularly when the collagen is atelopeptide collagen. Atelopeptide collagen is produced by pepsin-solubilization of native collagen. Since pepsin cuts off the telopeptides of the collagen molecule which are antigenic, pepsin-solubilized collagen is the most utilized form of collagen in the biomedical industry.
In the method disclosed by Brodsky et al in U.S. Pat. No. 4,971,954, the cross-linking occurs by a process of glycation. In this process the acyclic form of D(xe2x88x92)Ribose condenses spontaneously with the xcex5-amino groups of lysyl and hydroxylysyl residues located in the triple helical domain of the collagen molecule. The condensation product is a Schiff base that undergoes Amadori rearrangement to form a ketoamine adduct. Ketoamines located on adjacent collagen molecules condense to form covalent cross-links, the exact nature of which has yet not been determined, even though fluorescent heterocyclic structures and others type have been recently proposed.
Brodsky et al. disclose the process of glycation for native fibrillar type I collagen, such as for example the native fibrillar type I collagen from rat tendon. However, cross-linking by the glycation method of Brodsky et al. is reversible. For example, in an article entitled xe2x80x9cISOLATION AND PARTIAL CHARACTERIZATION OF COLLAGEN CHAINS DIMERIZED BY SUGAR-DERIVED CROSS-LINKSxe2x80x9d, published in The Journal of Biological Chemistry Vol. 263(33), pp. 17650-17657, 1988, Tanaka et al. show that rat tendon collagen cross-linked with D(xe2x88x92)Ribose for 1 day, is in the range of 50% reversibility at the end of a period of 5 days.
U.S. Pat. No. 5,955,438 to Pitaru et al. discloses, inter alia, a method for preparation of collagen matrices and membranes made from atelopeptide reconstituted collagen fibrils formed into a membrane and then cross-linked by a reducing sugar such as D(xe2x88x92)Ribose. The membrane or the implants made thereof are then subjected to critical point drying for drying and sterilization while preserving the three dimensional shape of the implants. The critical point drying procedure improves the resistance of the collagen matrix to collagenase degradation.
The cross-linking of native collagen with D(xe2x88x92)Ribose renders the native collagen fibers resistant to collagenase degradation. However, cross-linking of atelopeptide reconstituted collagen fibrils by D(xe2x88x92)Ribose is only negligibly effective in increasing their resistance to collagenase degradation. The reason for this is not clear. Since work by Tanaka et al. (see reference list) indicates that ribose-induced cross-links between native collagen molecules occur mainly between the triple-helical portions of adjacent collagen molecules, the removal of the telopeptides should not affect the degree of cross-linking of atelopeptide collagen. One possible explanation is that the packing of the atelopeptide collagen molecules in reconstituted collagen fibrils differs from the packing in native collagen fibrils (as discussed in Ref. 16 of the reference list). This difference in packing, in turn, may result in a change in the intermolecular distance or alignment which may cause a decrease in the strength or number of the covalent cross-links formed by D(xe2x88x92)Ribose.
There is therefore provided, in accordance with a preferred embodiment of the present invention, a method for preparing cross-linked collagen. The method includes the step of incubating collagen in a solution including water, at least one polar solvent, and at least one sugar, to form cross-linked collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the sugar is a reducing sugar.
Furthermore, in accordance with another preferred embodiment of the present invention, the polar solvent is an organic polar solvent.
Furthermore, in accordance with another preferred embodiment of the present invention, the organic polar solvent is an alcohol.
Furthermore, in accordance with another preferred embodiment of the present invention, the organic polar solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol acetone, tetrahydrofuran, dimethylsulfoxide, and combinations thereof.
Furthermore, in accordance with another preferred embodiment of the present invention, the polar solvent is miscible in water.
Furthermore, in accordance with another preferred embodiment of the present invention, the solution is a buffered solution including a buffer.
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes phosphate buffered saline.
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes water in the range of 15%-95% (v/v), at least one polar solvent in the range of 5%-85% (v/v), and a buffer.
Furthermore, in accordance with another preferred embodiment of the present invention, the collagen is selected from, native collagen, fibrillar collagen, fibrillar atelopeptide collagen, lyophylized collagen, collagen obtained from animal sources, human collagen, recombinant collagen, pepsinized collagen, reconstituted collagen, and combinations thereof.
Furthermore, in accordance with another preferred embodiment of the present invention, the collagen comprises fibrillar collagen reconstituted from monomolecular atelopeptide collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the collagen is obtained by reconstituting monomolecular atelopeptide collagen obtained by proteolytic digestion of native collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the sugar is a compound represented by one of the following formulae I or II: 
wherein:
R1 is H or lower alkyl or alkylene, an amino acid, a peptide, a saccharide, a purine or a pyrimidine base, a phosphorylated purine or pyrimidine base;
n is an integer between 2-9, and
p and q are each independently an integer between 0-8, and the sum of p and q is at least 2 and not more than 8.
Furthermore, in accordance with another preferred embodiment of the present invention, the sugar is a naturally occurring reducing sugar.
Furthermore, in accordance with another preferred embodiment of the present invention, the sugar is a diose, a triose, a tetrose, a pentose, a hexose, a septose, an octose, a nanose, or a decose.
Furthermore, in accordance with another preferred embodiment of the present invention, the sugar is selected from the group consisting of glycerose, threose, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose.
Furthermore, in accordance with another preferred embodiment of the present invention, the sugar is a disaccharide.
Furthermore, in accordance with another preferred embodiment of the present invention, the disaccharide is selected from the group consisting of maltose, lactose, sucrose, cellobiose, gentiobiose, melibiose, turanose, and trehalose.
Furthermore, in accordance with another preferred embodiment of the present invention, at least one substance is added to the solution in which the step of incubating is performed, the substance becoming immobilized within the matrix.
Furthermore, in accordance with another preferred embodiment of the present invention, the substance is selected from the group consisting of an antimicrobial agent, an anti-inflammatory agent, a factor having tissue inductive properties, and combinations thereof.
Furthermore, in accordance with another preferred embodiment of the present invention, the sugar is D(xe2x88x92)ribose, and the polar solvent is ethanol.
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes water in the range of 15%-95% (v/v) and ethanol in the range of 5%-85% (v/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes water in the range of 25%-50% (v/v) and ethanol in the range of 50%-75% (v/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes about 30% water (v/v), and about 70% ethanol (v/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the concentration of D(xe2x88x92)ribose in the solution is in the range of 0.1%-5% (w/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the concentration of D(xe2x88x92)ribose in the solution is in the range of 0.5%-3% (w/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of washing the cross-linked collagen after the step of incubating, to remove the polar solvent and excess of the sugar.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of dehydrating the cross-linked collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of subjecting the cross-linked collagen to critical point drying.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of drying or freeze-drying the collagen prior to the step of incubating.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of drying or freeze-drying the cross-linked collagen.
There is also provided, in accordance with another preferred embodiment of the present invention, a cross-linked collagen preparation prepared by the method disclosed hereinabove.
There is also provided, in accordance with another preferred embodiment of the present invention, a method for preparing cross-linked collagen. The method includes the step of incubating collagen in a solution including water, at least one hydrophilic solvent and at least one sugar to form the cross-linked collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of controlling the duration of the incubating of the step of incubating to control the degree of cross linking of the cross-linked collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of controlling the concentration of the sugar used in the step of incubating to control the degree of cross linking of the cross-linked collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of controlling the concentration of the hydrophilic solvent used in the step of incubating to control the degree of cross linking of the cross-linked collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of removing at least some of the unreacted amount of the sugar, and removing at least some of the hydrophilic solvent.
Furthermore, in accordance with another preferred embodiment of the present invention, the method further includes the step of washing the cross-linked collagen to remove at least some of the unreacted amount of the sugar and to remove at least some of the hydrophilic solvent.
There is also provided, in accordance with another preferred embodiment of the present invention, a method for preparing cross-linked collagen. The method includes the step of incubating collagen in a solution including water, at least one polar solvent and D(xe2x88x92)Ribose.
Furthermore, in accordance with another preferred embodiment of the present invention, the polar solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol acetone, tetrahydrofuran, dimethylsulfoxide, and combinations thereof.
Furthermore, in accordance with another preferred embodiment of the present invention, the collagen is selected from, native collagen, fibrillar collagen, fibrillar atelopeptide collagen, lyophylized collagen, collagen obtained from animal sources, human collagen, recombinant collagen, pepsinized collagen, reconstituted collagen, and combinations thereof.
Furthermore, in accordance with another preferred embodiment of the present invention, the collagen includes fibrillar collagen reconstituted from monomolecular atelopeptide collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the collagen is atelopeptide fibrillar collagen obtained by reconstituting monomolecular atelopeptide collagen obtained by proteolytic digestion of native collagen.
Furthermore, in accordance with another preferred embodiment of the present invention, the concentration of D(xe2x88x92)ribose in the solution is in the range of 0.1%-5% (w/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the concentration of D(xe2x88x92)ribose in the solution is in the range of 0.5%-3% (w/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes water in the range of 15%-95% (v/v) and at least one polar solvent in the range of 5%-85% (v/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes phosphate buffered saline in the range of 15%-95% (v/v) and at least one polar solvent in the range of 5%-85% (v/v).
Furthermore, in accordance with another preferred embodiment of the present invention, the solution is a buffered solution including a buffer.
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes phosphate buffered saline.
Furthermore, in accordance with another preferred embodiment of the present invention, the solution includes water in the range of 15%-95% (v/v), at least one polar solvent in the range of 5%-85% (v/v), and a buffer.
There is also provided, in accordance with another preferred embodiment of the present invention, a method for preparing cross-linked collagen, the method includes the step of incubating reconstituted atelopeptide fibrillar collagen in a solution including water, at least one polar solvent and at least one reducing sugar.
Furthermore, in accordance with another preferred embodiment of the present invention, solution is a buffered solution.
There is also provided, in accordance with another preferred embodiment of the present invention, a method for preparing a cross-linked collagen having a desired resistance to degradation. The method includes the steps of incubating collagen in a solution including water, at least one polar solvent, and at least one sugar, and controlling the duration of incubating the collagen to obtain cross-linked collagen having a desired resistance to degradation.
There is also provided, in accordance with another preferred embodiment of the present invention, a method for preparing a cross-linked collagen having a desired resistance to degradation. The method includes the steps of incubating collagen in a solution including water, at least one polar solvent, and at least one sugar, and selecting the concentration of the polar solvent to obtain cross-linked collagen having a desired resistance to degradation.
There is also provided, in accordance with another preferred embodiment of the present invention, a method for preparing a cross-linked collagen having a desired resistance to degradation. The method includes the steps of incubating collagen in a solution including water, at least one polar solvent and at least one sugar, and selecting the concentration of the sugar used in the step of incubating to obtain cross-linked collagen having a desired resistance to degradation.
There is also provided, in accordance with another preferred embodiment of the present invention, an improved cross-linked fibrillar collagen matrix obtained by a process for its preparation from fibrillar collagen. The process includes the steps of providing a matrix including reconstituted fibrillar collagen, and incubating the matrix in a solution including water, at least one polar solvent and at least one sugar, for cross-linking the fibrillar collagen to form a cross-linked fibrillar collagen matrix.
Furthermore, in accordance with another preferred embodiment of the present invention, the matrix is in the form of an implantable device.
Furthermore, in accordance with another preferred embodiment of the present invention, the implantable device is a collagen based membrane barrier for guided tissue regeneration.
Furthermore, in accordance with another preferred embodiment of the present invention, the process used for preparing the matrix further includes the step of washing the cross-linked collagen matrix after the step of incubating to remove at least some of the polar solvent and unreacted excess of the sugar.
Furthermore, in accordance with another preferred embodiment of the present invention, the process further includes the step of dehydrating the cross-linked fibrillar collagen matrix.
Furthermore, in accordance with another preferred embodiment of the present invention, the process used for preparing the matrix further includes the step of subjecting the cross-linked fibrillar collagen matrix to critical point drying.
Furthermore, in accordance with another preferred embodiment of the present invention, the process used for preparing the matrix further includes the step of drying or freeze-drying the cross-linked fibrillar collagen matrix.
Furthermore, in accordance with another preferred embodiment of the present invention, the fibrillar collagen comprises fibrillar collagen reconstituted from monomolecular atelopeptide collagen.
Finally, in accordance with another preferred embodiment of the present invention, the fibrillar collagen is prepared by reconstituting monomolecular atelopeptide collagen obtained by proteolytic digestion of native collagen.